Medium for selectively growing specific bacteria of genus bifidobacterium

Abstract

To provide a technology for selectively growing specific bacteria of the genus Bifidobacterium.SOLUTION: A medium for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621, the medium comprising lactose and used at 38°C to 45°C at the time of use.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a culture medium for selectively growing specific Bifidobacterium bacteria. [Background technology]

[0002] In research on the intestinal flora or research using fecal samples, it is sometimes necessary to selectively grow specific Bifidobacterium bacteria without growing other bacteria.

[0003] In order to selectively grow specific Bifidobacterium bacteria, a selective medium for Bifidobacterium bacteria has been developed, which is characterized by being a completely synthetic medium containing, for example, one or more sugars selected from the group consisting of glucose, lactose, and fructose, an ammonium salt, biotin, pantothenic acid, riboflavin, pyruvic acid, polyoxyethylene sorbitan mono-fatty acid ester, and nalidixic acid (Patent Document 1).

[0004] In addition, a method has been developed for measuring the viable cell count of only microorganisms belonging to Bifidobacterium longum subsp. longum from test bacteria containing microorganisms belonging to the genus Bifidobacterium using a culture medium (Patent Document 2). Specifically, the test bacteria include microorganisms belonging to the genus Bifidobacterium, such as microorganisms belonging to Bifidobacterium longum subsp. longum, and a specified microorganism; the measurement of the viable cell count is characterized by culturing the test bacteria in the culture medium under anaerobic conditions at 37°C for 48 hours, and then counting colonies with a diameter of 0.7 mm or more formed in the culture medium; and the culture medium satisfies the following 1) to 5). 1) Contains only L-arabinose as a sugar source; 2) The concentration of L-arabinose in the medium is 2 to 3% by mass based on the total mass of the medium. 3) containing peptone, meat extract, and yeast extract as nitrogen sources; 4) The content of the peptone is 6.0 to 14.0 g / 1000 mL, the content of the meat extract is 6.0 to 14.0 g / 1000 mL, and the content of the yeast extract is 1.8 to 4.2 g / 1000 mL. 5) Contains neither magnesium sulfate nor manganese sulfate.

[0005] Furthermore, TOS propionic acid agar medium is commercially available as a medium for measuring the viable count of Bifidobacterium bacteria (Non-Patent Document 1, Yakult Pharmaceutical Co., Ltd.). The ingredients of this medium per 1,000 mL of medium are 10 g of peptone, 1.0 g of yeast extract, 3.0 g of potassium dihydrogen phosphate, 4.8 g of dipotassium monohydrogen phosphate, 3.0 g of ammonium sulfate, 0.2 g of magnesium sulfate (heptahydrate), 0.5 g of L-cysteine ​​hydrochloride (monohydrate), 15 g of sodium propionate, 10 g of galactooligosaccharide, and 15 g of agar (pH is approximately 6.3). A method for measuring the viable cell count using this medium is described as follows: 62.5 g of medium is dissolved by heating in 1,000 ml of purified water, and then the viable cell count is measured after sterilization at 115°C for 15 minutes (Non-Patent Document 1). To measure the viable cell count, the medium is kept at approximately 45°C and mixed with a certain amount of test material, or the medium is spread on a petri dish in advance, dried, and then a certain amount is smeared on the surface. In either case, the medium is then anaerobic cultured at 37°C for 72 hours before measurement. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 63-066518 [Patent Document 2] Patent No. 5401558 [Non-patent literature]

[0007] [Non-Patent Document 1] Yakult Pharmaceutical Co., Ltd., "TOS Propionic Acid Agar Medium", [Retrieved March 12, 2024], Internet,<URL:https: / / www.yakult.co.jp / ypi / product / tos.html> Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide a technology for selectively growing specific Bifidobacterium bacteria, specifically, Bifidobacterium longum subsp. longum NITE BP-02621. [Means for solving the problem]

[0009] The present inventors have found that the above problems can be solved by focusing on the medium composition and culture temperature, and have completed the present invention.

[0010] The present invention provides A medium for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621, Contains lactose, Use at 38-45°C. A culture medium can be provided.

[0011] The present invention also provides A method for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621, comprising: inoculating the sample into a medium containing lactose; and Cultivating the bacteria contained in the sample at 38 to 45°C A method can be provided, comprising:

[0012] The present invention also provides A medium for detecting Bifidobacterium longum subsp. longum NITE BP-02621, Contains lactose, Use at 38-45°C. A culture medium can be provided.

[0013] The present invention also provides A method for detecting Bifidobacterium longum subsp. longum NITE BP-02621, comprising: inoculating the sample into a medium containing lactose; Culturing the bacteria contained in the sample at 38 to 45°C; and detecting colonies after the culturing step A method can be provided, comprising: [Effects of the Invention]

[0014] According to the present invention, a technique for selectively growing specific Bifidobacterium bacteria can be provided. Specifically, Bifidobacterium longum subsp. It is possible to provide a technology for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621. This technology is useful when it is desired to selectively grow a specific bacterium in research on the intestinal flora or research using fecal samples, etc. Furthermore, because it is possible to selectively grow a specific bacterium, it can also provide a technology for detecting the specific bacterium from a sample containing multiple species of bacteria. DETAILED DESCRIPTION OF THE INVENTION

[0015] In this specification, Bifidobacterium longum subsp. longum NITE BP-02621 may be referred to as the "bacterium of the present invention" or the "strain of the present invention." Bifidobacterium longum subsp. longum is sometimes simply referred to as Bifidobacterium longum, and Bifidobacterium longum subsp. infantis is sometimes simply referred to as Bifidobacterium infantis.

[0016] (Culture medium for selectively growing specific Bifidobacterium bacteria) A first aspect of the present invention is A medium for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621, Contains lactose, Use at 38-45°C. Culture medium is.

[0017] Bifidobacterium longum subsp. longum NITE BP-02621 is identical to Bifidobacterium longum BB536 (NITE BP-02621). This strain was internationally deposited on January 26, 2018, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02621.

[0018] The strain of the present invention is not limited to the strain deposited or registered with a designated institution under that name (hereinafter, for convenience of explanation, also referred to as the "deposited strain"), but also includes strains that are substantially equivalent to the deposited strain (hereinafter, also referred to as "derived strains"). In other words, "Bifidobacterium longum BB536 (NITE BP-02621)" is not limited to the strain deposited with the above depository institution under the accession number NITE BP-02621, but also includes strains that are substantially equivalent to the deposited strain. A "strain substantially equivalent to the deposited strain" refers to a strain that belongs to the same species as the deposited strain, can be selectively grown equivalently to NITE BP-02621, has a nucleotide sequence of its 16S rRNA gene that is preferably 99.86% or more, more preferably 99.93% or more, and even more preferably 100% identical to the nucleotide sequence of the 16S rRNA gene of the deposited strain, and preferably has the same biological properties as the deposited strain. A strain substantially equivalent to the deposited strain may be, for example, a derivative strain obtained using the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that have naturally arisen from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), etc.). Strains naturally derived from the deposited strain include strains naturally derived during use of the deposited strain. Use of the deposited strain includes culturing (e.g., subculturing) the deposited strain. Derivative strains may be constructed by one type of modification, or by two or more types of modifications.

[0019] The medium of this embodiment is solid (including gel-like) when used. Therefore, the medium of this embodiment contains a component that becomes solid (including gel-like) when used. Such a component can be, for example, a gelling agent. A gelling agent is a substance that swells and gels when it absorbs water, and acts as a matrix for forming the medium.

[0020] The form of the medium of this embodiment when used is not limited, but may be, for example, solidified in a container such as a petri dish, which is preferred because it allows the bacteria of the present invention to grow as colonies and be clearly detected.

[0021] The gelling agent may be any of those used in conventional solid media for culturing the bacterium of the present invention. Examples include polymeric compounds (e.g., thickening polysaccharides, water-absorbent polymers, etc.). Specific examples include agar, guar gum, xanthan gum, locust bean gum, gellan gum, polyvinyl alcohol, alkyl celluloses (e.g., methyl cellulose, ethyl cellulose, etc.), carboxyalkyl celluloses (e.g., carboxymethyl cellulose, carboxyethyl cellulose, etc.), and hydroxyalkyl celluloses (e.g., hydroxymethyl cellulose, hydroxyethyl cellulose, etc.). One or more of these may be used. Furthermore, the average molecular weight, degree of polymerization, etc. may be within the ranges of those used in conventional solid media for culturing the bacterium of the present invention.

[0022] When the gelling agent is agar, the content of agar in the medium of this embodiment is not limited as long as the medium of this embodiment is easy to handle and mold, similar to conventional solid media used to culture the bacterium of the present invention. The concentration may be, for example, 4.5 g / L or more, 7.5 g / L or more, 12 g / L or more, 15 g / L or more, 18 g / L or more, 23 g / L or more, or, for example, 30 g / L or less, 23 g / L or less, 18 g / L or less, 15 g / L or less, 12 g / L or less, 7.5 g / L or less. Consistent combinations thereof are also acceptable. For example, 4.5 to 7.5 g / L, 7.5 to 12 g / L, 12 to 15 g / L, 15 to 18 g / L, 18 to 23 g / L, 23 to 30 g / L, etc.

[0023] The medium of this embodiment contains a sugar. The sugar is primarily lactose, but other sugars may be contained in addition to lactose. Examples of other sugars include glucose, sucrose, xylose, cellobiose, maltose, and galactooligosaccharides. One or more of these may be used.

[0024] The lactose content relative to the total amount of sugars contained in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but may be, for example, 1% by mass or more, 5% by mass or more, 10% by mass or more, 30% by mass or more, 50% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, etc. On the other hand, a higher upper limit is preferable, for example, 100% by mass or less, 99% by mass or less, 98% by mass or less, etc. Consistent combinations thereof are also acceptable, for example, 1 to 100% by mass, 5 to 100% by mass, 10 to 100% by mass, 30 to 99% by mass, 50 to 99% by mass, 70 to 99% by mass, 80 to 98% by mass, 90 to 98% by mass, 95 to 98% by mass, etc. Alternatively, it may be 100% by mass.

[0025] The lactose content in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but may be, for example, 3.0 g / L or more, 5.0 g / L or more, 8.0 g / L or more, 10 g / L or more, 12 g / L or more, 15 g / L or more, or, for example, 20 g / L or less, 15 g / L or less, 12 g / L or less, 10 g / L or less, 8.0 g / L or less, 5.0 g / L or less, or any combination thereof that does not contradict these. For example, 3.0 to 5.0 g / L, 5.0 to 8.0 g / L, 8.0 to 10 g / L, 10 to 12 g / L, 12 to 15 g / L, 15 to 20 g / L, or the like. When the lactose content in the medium of this embodiment is above the lower limit and below the upper limit, it is presumed that the bacterium of the present invention will selectively grow because it utilizes lactose as a sugar source and acquires heat resistance by, for example, changing gene expression, and / or because the bacterium of the present invention utilizes lactose as a sugar source under stressful environments such as high temperatures, and therefore this is preferred.

[0026] The medium of this embodiment may contain components other than those described above, as long as the effects of the present invention are achieved. For example, it may contain components contained in a typical solid medium used to culture the bacterium of the present invention. Furthermore, the content of each component in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, and may be, for example, the content contained in a typical solid medium used to culture the bacterium of the present invention.

[0027] Examples include peptone (e.g., casein enzyme hydrolysate, etc.), yeast extract, meat extract, fish meat extract, potassium dihydrogen phosphate, dipotassium monohydrogen phosphate, ammonium sulfate, magnesium sulfate (heptahydrate), L-cysteine ​​hydrochloride (monohydrate), sodium chloride, disodium hydrogen phosphate, magnesium sulfate, sodium thiosulfate, sodium propionate, sodium pyruvate, ammonium ferric citrate, sodium citrate, starch, starch derivatives, hyaluronic acid, acrylic acid derivatives, polyether, collagen, sodium carbonate, sodium bicarbonate, etc. One or more of these may be contained. The enzymatic casein hydrolysate can be prepared according to a conventional method, or a commercially available product such as Bacto (registered trademark) Proteose Peptone No. 3 (Gibco (registered trademark), product number (catalog number): 211693) can be used.

[0028] Examples of antibiotics include trimethoprim, mupirocin, polylysine, protamine sulfate, glycine, sorbic acid, etc. One or more of these may be contained.

[0029] The content of peptone (e.g., enzymatic casein hydrolysate, etc.) in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but the concentration may be, for example, 3.0 g / L or more, 5.0 g / L or more, 8.0 g / L or more, 10 g / L or more, 12 g / L or more, 15 g / L or more, etc., or, for example, 20 g / L or less, 15 g / L or less, 12 g / L or less, 10 g / L or less, 8.0 g / L or less, 5.0 g / L or less, etc. Consistent combinations thereof are also acceptable, such as 3.0 to 5.0 g / L, 5.0 to 8.0 g / L, 8.0 to 10 g / L, 10 to 12 g / L, 12 to 15 g / L, 15 to 20 g / L, etc.

[0030] The content of yeast extract in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but the concentration may be, for example, 0.30 g / L or more, 0.50 g / L or more, 0.80 g / L or more, 1.0 g / L or more, 1.2 g / L or more, 1.5 g / L or more, or, for example, 2.0 g / L or less, 1.5 g / L or less, 1.2 g / L or less, 1.0 g / L or less, 0.80 g / L or less, 0.50 g / L or less, or any combination thereof that does not contradict these. For example, 0.30 to 0.50 g / L, 0.50 to 0.80 g / L, 0.80 to 1.0 g / L, 1.0 to 1.2 g / L, 1.2 to 1.5 g / L, 1.5 to 2.0 g / L, or the like.

[0031] The content of potassium dihydrogen phosphate in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but may be, for example, 0.90 g / L or more, 1.5 g / L or more, 2.4 g / L or more, 3.0 g / L or more, 3.6 g / L or more, 4.5 g / L or more, or, for example, 6.0 g / L or less, 4.5 g / L or less, 3.6 g / L or less, 3.0 g / L or less, 2.4 g / L or less, 1.5 g / L or less, or any combination thereof that does not contradict these. For example, 0.90 to 1.5 g / L, 1.5 to 2.4 g / L, 2.4 to 3.0 g / L, 3.0 to 3.6 g / L, 3.6 to 4.5 g / L, 4.5 to 6.0 g / L, or the like.

[0032] The content of dipotassium monohydrogen phosphate in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but the concentration may be, for example, 1.4 g / L or more, 2.4 g / L or more, 3.8 g / L or more, 4.8 g / L or more, 5.8 g / L or more, 7.2 g / L or more, etc., or, for example, 9.6 g / L or less, 7.2 g / L or less, 5.8 g / L or less, 4.8 g / L or less, 3.8 g / L or less, 2.4 g / L or less, etc. Consistent combinations thereof are also acceptable, such as 1.4 to 2.4 g / L, 2.4 to 3.8 g / L, 3.8 to 4.8 g / L, 4.8 to 5.8 g / L, 5.8 to 7.2 g / L, 7.2 to 9.6 g / L, etc.

[0033] The ammonium sulfate content in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but may be, for example, 0.90 g / L or more, 1.5 g / L or more, 2.4 g / L or more, 3.0 g / L or more, 3.6 g / L or more, 4.5 g / L or more, or, for example, 6.0 g / L or less, 4.5 g / L or less, 3.6 g / L or less, 3.0 g / L or less, 2.4 g / L or less, 1.5 g / L or less, or any combination thereof that does not contradict these. For example, 0.90 to 1.5 g / L, 1.5 to 2.4 g / L, 2.4 to 3.0 g / L, 3.0 to 3.6 g / L, 3.6 to 4.5 g / L, 4.5 to 6.0 g / L, or the like.

[0034] The content of magnesium sulfate (heptahydrate) in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but the concentration may be, for example, 0.060 g / L or more, 0.10 g / L or more, 0.16 g / L or more, 0.20 g / L or more, 0.24 g / L or more, 0.30 g / L or more, or, for example, 0.40 g / L or less, 0.30 g / L or less, 0.24 g / L or less, 0.20 g / L or less, 0.16 g / L or less, 0.10 g / L or less, or any combination thereof that does not contradict these. For example, 0.060 to 0.10 g / L, 0.10 to 0.16 g / L, 0.16 to 0.20 g / L, 0.20 to 0.24 g / L, 0.24 to 0.30 g / L, 0.30 to 0.40 g / L, or the like.

[0035] The content of L-cysteine ​​hydrochloride (monohydrate) in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but may be, for example, 0.15 g / L or more, 0.25 g / L or more, 0.40 g / L or more, 0.50 g / L or more, 0.60 g / L or more, or 0.75 g / L or more, or, for example, 1.0 g / L or less, 0.75 g / L or less, 0.60 g / L or less, 0.50 g / L or less, 0.40 g / L or less, or 0.25 g / L or less. Consistent combinations thereof are also acceptable, such as 0.15 to 0.25 g / L, 0.25 to 0.40 g / L, 0.40 to 0.50 g / L, 0.50 to 0.60 g / L, 0.60 to 0.75 g / L, or 0.75 to 1.0 g / L.

[0036] The content of sodium propionate in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but may be, for example, 4.5 g / L or more, 7.5 g / L or more, 12 g / L or more, 15 g / L or more, 18 g / L or more, 23 g / L or more, or, for example, 30 g / L or less, 23 g / L or less, 18 g / L or less, 15 g / L or less, 12 g / L or less, 7.5 g / L or less, or any combination thereof that does not contradict these. For example, 4.5 to 7.5 g / L, 7.5 to 12 g / L, 12 to 15 g / L, 15 to 18 g / L, 18 to 23 g / L, 23 to 30 g / L, or the like.

[0037] The content of trimethoprim in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but the concentration may be, for example, 0.003 g / L or more, 0.004 g / L or more, 0.006 g / L or more, or, for example, 0.020 g / L or less, 0.012 g / L or less, 0.010 g / L or less, or any combination thereof that does not contradict these. For example, 0.003 to 0.010 g / L, 0.004 to 0.012 g / L, 0.006 to 0.020 g / L, or the like.

[0038] The mupirocin content in the medium of this embodiment is not limited as long as the effects of the present invention are achieved, but may be, for example, 0.01 g / L or more, 0.02 g / L or more, 0.03 g / L or more, or, for example, 0.2 g / L or less, 0.15 g / L or less, 0.1 g / L or less, or any combination thereof that does not contradict these. For example, 0.01 to 0.1 g / L, 0.02 to 0.15 g / L, 0.03 to 0.2 g / L, etc.

[0039] The pH of the medium in this embodiment may be the pH of a typical solid medium used to culture the bacterium of the present invention, such as 6.0 or higher, 6.1 or higher, 6.2 or higher, or 6.3 or higher, or may be 6.6 or lower, 6.5 or lower, 6.4 or lower, or 6.3 or lower, or any compatible combination thereof, such as 6.0 to 6.3, 6.1 to 6.4, 6.2 to 6.5, or 6.3 to 6.6.

[0040] The medium of this embodiment is a medium for selectively growing the bacterium of the present invention. "Selectively growing the bacteria of the present invention" may mean selectively growing the bacteria of the present invention without growing other bacteria. The other bacteria can be contained in a sample to be inoculated into the medium of this embodiment, and are not limited as long as they do not grow when the medium of this embodiment is used. In other words, the medium of this embodiment selectively grows the bacteria of the present invention without growing other bacteria. The medium may be a medium for culturing the cells.

[0041] The medium of this embodiment allows the bacteria of the present invention to selectively grow as colonies and be clearly detected. Therefore, "selectively growing the bacteria of the present invention" may mean selectively growing the bacteria of the present invention as colonies. Furthermore, "selectively growing the bacteria of the present invention without allowing other bacteria to grow" may mean selectively growing the bacteria of the present invention as colonies while not allowing other bacteria to grow as colonies.

[0042] The sample to be inoculated into the medium of this embodiment may be a sample containing the bacterium of the present invention and the other bacteria as bacteria. The sample to be inoculated into the medium of this embodiment may be a specimen or a processed specimen (e.g., a specimen dilution obtained by diluting a specimen, a specimen concentrate obtained by concentrating a specimen, etc.). Examples of specimens include feces, digestive tract contents, oral fluids, foods and beverages (e.g., supplements, bacterial powder products, yogurt, fermented vinegar, oil drops, and modified powdered milk), and pharmaceuticals. That is, the sample to be inoculated into the culture medium of this embodiment may be a specimen containing the bacterium of the present invention and the other bacteria as bacteria, or may be a processed specimen.

[0043] Examples of the other bacteria include enterobacteria (excluding the bacteria of the present invention). Specific examples include bacteria of the genus Klebsiella, Rothia, Bifidobacterium (excluding the bacteria of the present invention), Enterococcus, Streptococcus, Escherichia, Staphylococcus, Lactobacillus, Turicibacter, Clostridium, Ruminococcus, Veillonella, Bacteroides, Parabacteroides, and Lactococcus.

[0044] Examples of bacteria of the genus Bifidobacterium (excluding the bacteria of the present invention) include Bifidobacterium longum (excluding the bacteria of the present invention), such as Bifidobacterium longum subsp. longum (excluding the bacteria of the present invention), Bifidobacterium longum subsp. infantis, and Bifidobacterium longum subsp. suis. Another example is Bifidobacterium breve. Further examples include Bifidobacterium animalis, such as Bifidobacterium animalis subsp. lactis and Bifidobacterium animalis subsp. animalis. Other examples include Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, and Bifidobacterium pseudocatenulatum. Further examples include Bifidobacterium pseudolongum, such as Bifidobacterium pseudolongum subsp. globosum and Bifidobacterium pseudolongum subsp. pseudolongm. Other examples include Bifidobacterium thermophilum.

[0045] Examples of Bifidobacterium longum subsp. longum include JCM 1217 T , ATCC 15708, ATCC 51870, NITE BP-02497, NITE BP-02568, NITE BP-02564, NITE BP-02565, NITE BP-02569, NITE BP-02572, NITE BP-02499, NITE BP-02574, NITE BP-02566, NITE BP-02706, NITE BP-02567, NITE BP-02500, NITE BP-02430, NITE BP-03751, and the like.

[0046] Bacteria assigned JCM numbers can be obtained from the Japan Collection of Microorganisms (JCM, postal code: 305-0074, address: Microbial Materials Development Division, RIKEN BioResource Research Center, 3-1-1 Takanodai, Tsukuba, Ibaraki, Japan) or from the depository institutions where the respective strains have been deposited. Bacteria assigned ATCC numbers can be obtained from the American Type Culture Collection (ATCC, Address: 10801 University Boulevard, Manassas, VA 20110, United States of America) or from the depository institution where each strain has been deposited.

[0047] Bifidobacterium longum subsp. longum NITE BP-02497 is identical to Bifidobacterium longum MCC0300 (NITE BP-02497). This strain was internationally deposited on June 22, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02497.

[0048] Bifidobacterium longum subsp. longum NITE BP-02568 is identical to Bifidobacterium longum MCLON2FL1 (NITE BP-02568). This strain was internationally deposited on November 10, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02568.

[0049] Bifidobacterium longum subsp. longum NITE BP-02564 is identical to Bifidobacterium longum MCLONSIAL1 (NITE BP-02564). This strain was deposited on November 10, 2017, with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (ROI), 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818, Address: Room 122) under the accession number NITE P-02564. On October 9, 2018, it was transferred to international deposit under the Budapest Treaty and assigned the accession number NITE BP-02564.

[0050] Bifidobacterium longum subsp. longum NITE BP-02565 is identical to Bifidobacterium longum MCLONSIAL2 (NITE BP-02565). This strain was deposited on November 10, 2017, with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (ROI), 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818, Address: Room 122) under the accession number NITE P-02565. On October 9, 2018, it was transferred to international deposit under the Budapest Treaty and assigned the accession number NITE BP-02565.

[0051] Bifidobacterium longum subsp. longum NITE BP-02569 is identical to Bifidobacterium longum MCLON2FL2 (NITE BP-02569). This strain was identified as Bifidobacterium longum MCLON2FL2 (NITE BP-02569) on November 10, 2017. The strain has been internationally deposited with the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) based on the Budapest Treaty, and has been assigned the accession number NITE BP-02569.

[0052] Bifidobacterium longum subsp. longum NITE BP-02572 is identical to Bifidobacterium longum McLonenda1 (NITE BP-02572). This strain was deposited under the Budapest Treaty on November 13, 2017, with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (ROI), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818). The strain has been assigned the accession number NITE BP-02572.

[0053] Bifidobacterium longum subsp. longum NITE BP-02499 is identical to Bifidobacterium longum MCC10085 (NITE BP-02499). This strain was internationally deposited on June 22, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02499.

[0054] Bifidobacterium longum subsp. longum NITE BP-02574 is identical to Bifidobacterium longum McLonenda3 (NITE BP-02574). This strain was internationally deposited on November 13, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (ROI), 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code: 292-0818, Address: Room 122), and has been assigned the accession number NITE BP-02574.

[0055] Bifidobacterium longum subsp. longum NITE BP-02566 is identical to Bifidobacterium longum McLonpull1 (NITE BP-02566). This strain was internationally deposited on November 10, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02566.

[0056] Bifidobacterium longum subsp. longum NITE BP-02706 is identical to Bifidobacterium longum MCLONBATH2 (NITE BP-02706). This strain was internationally deposited on May 7, 2018, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02706.

[0057] Bifidobacterium longum subsp. longum NITE BP-02567 is identical to Bifidobacterium longum McLonpull2 (NITE BP-02567). This strain was internationally deposited on November 10, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02567.

[0058] Bifidobacterium longum subsp. longum NITE BP-02500 is identical to Bifidobacterium longum MCC10127 (NITE BP-02500). This strain was internationally deposited on June 22, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02500.

[0059] Bifidobacterium longum subsp. longum NITE BP-02430 is identical to Bifidobacterium longum MCC1110 (NITE BP-02430). This strain was internationally deposited on February 21, 2017, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-02430.

[0060] Bifidobacterium longum subsp. longum NITE BP-03751 is identical to Bifidobacterium longum MCC10345 (NITE BP-03751). This strain was internationally deposited on September 14, 2022, under the Budapest Treaty with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and has been assigned the accession number NITE BP-03751.

[0061] The strain is not limited to the strain deposited or registered under that name at a designated institution (hereinafter, for convenience of explanation, also referred to as the "deposited strain"), but also includes strains that are substantially equivalent to the deposited strain (hereinafter, also referred to as the "derived strain"). That is, Bifidobacterium longum subsp. longum JCM 1217 T For example, "Bifidobacterium longum subsp. longum JCM 1217 T " includes JCM 1217 TThe term "substantially equivalent to the deposited strain" is not limited to the strain itself deposited at the depository under the accession number, but also includes strains substantially equivalent to the deposited strain. A "strain substantially equivalent to the deposited strain" refers to a strain that belongs to the same species as the deposited strain, can be contained in a sample to be inoculated into the medium of this embodiment, does not grow when the medium is used, has a nucleotide sequence of its 16S rRNA gene that is preferably 99.86% or more, more preferably 99.93% or more, and even more preferably 100% identical to the nucleotide sequence of the 16S rRNA gene of the deposited strain, and preferably has the same biological properties as the deposited strain. A strain substantially equivalent to the deposited strain may be, for example, a derivative strain obtained using the deposited strain as a parent strain. Derivative strains include strains bred from the deposited strain and strains that naturally arise from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), etc.). Strains naturally derived from the deposited strain include strains naturally derived during use of the deposited strain. Use of the deposited strain includes culturing (e.g., subculturing) the deposited strain. Derivative strains may be constructed by one type of modification, or by two or more types of modifications.

[0062] The medium of this embodiment is used at a predetermined temperature. The predetermined temperature is, for example, 38°C or higher, 39°C or higher, 40°C or higher, 41°C or higher, 42°C or higher, 43°C or higher, 44°C or higher, etc., or, for example, 45°C or lower, 44°C or lower, 43°C or lower, etc. It may also be a compatible combination thereof. For example, 38 to 45°C, 39 to 44°C, 40 to 43°C, 41 to 44°C, 42 to 44°C, 43 to 45°C, 44 to 45°C, etc. When used at 38°C or higher, the bacteria of the present invention can be selectively grown, while when used at 45°C or lower, excessive killing of the bacteria of the present invention and excessive inhibition of growth of the bacteria of the present invention can be prevented.

[0063] The method for selectively growing the bacterium of the present invention using the medium of this embodiment will be described in the embodiment below.

[0064] When the medium of this embodiment is solidified in a container such as a petri dish before use, the bacteria of the present invention can be selectively grown as colonies. In this case, the diameter of the colonies may be a normal diameter when confirming colonies (e.g., a diameter that can be confirmed with the naked eye). Specifically, for example, the diameter may be 0.1 mm or more, 0.2 mm or more, 0.3 mm or more, or, for example, 10 mm or less, 8 mm or less, 5 mm or less, or any combination thereof that does not contradict these. For example, 0.1 to 10 mm, 0.2 to 8 mm, 0.3 to 5 mm, etc.

[0065] (Method for selectively growing specific Bifidobacterium bacteria) A second aspect of the present invention is A method for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621, comprising: inoculating the sample into a medium containing lactose; and Cultivating the bacteria contained in the sample at 38 to 45°C A method comprising: is.

[0066] With regard to Bifidobacterium longum subsp. longum NITE BP-02621, the explanation in the first embodiment is incorporated herein by reference.

[0067] This embodiment includes a step of inoculating a sample into a culture medium containing lactose (inoculation step).

[0068] For the lactose-containing medium in this step, the explanation of the medium in the first embodiment is applicable.

[0069] This embodiment is a method for selectively growing the bacterium of the present invention. "Selectively growing the bacterium of the present invention" may mean selectively growing the bacterium of the present invention without allowing other bacteria to grow. The other bacteria are not limited as long as they can be contained in a sample to be inoculated into a lactose-containing medium and do not grow in the culture step described below. In other words, the method of this embodiment may be a method of selectively growing the bacterium of the present invention without allowing other bacteria to grow.

[0070] According to the method of this embodiment, the bacterium of the present invention can be selectively grown as a colony and clearly detected. Therefore, "selectively growing the bacterium of the present invention" may mean selectively growing the bacterium of the present invention as a colony. Furthermore, "selectively growing the bacterium of the present invention without allowing other bacteria to grow" may mean selectively growing the bacterium of the present invention as a colony while not allowing other bacteria to grow as colonies.

[0071] The description of the sample in this step is the same as that in the first embodiment.

[0072] For other bacteria, the explanation in the first embodiment is applicable.

[0073] In this step, examples of the method for inoculating the lactose-containing medium with the sample include the pour plate method, the plate smear method, and the spiral method when the medium is in a solidified form in a container such as a petri dish at the time of use. The pour mixture method involves mixing the sample with the medium dissolved by heating, and then solidifying it by cooling (or at room temperature (about 25° C.)). The plate smear method is a method in which a sample is smeared on the medium. The spiral plating method involves plating a sample onto the medium in a concentration gradient.

[0074] This embodiment includes a step (cultivation step) of culturing the bacteria contained in the sample at a predetermined temperature (for example, 38 to 45° C.) after the inoculation step. The description of the first embodiment is applicable to the predetermined temperature. The culture method in this step may be a method commonly used when culturing the bacterium of the present invention. For example, anaerobic culture may be used. The culture time may also be a culture time commonly used when culturing the bacterium of the present invention. For example, it may be 48 hours or more, 60 hours or more, or 72 hours or more, or it may be 96 hours or less, 84 hours or less, or 72 hours or less. A compatible combination thereof may also be used. For example, it may be 48 to 72 hours, 60 to 84 hours, or 72 to 96 hours.

[0075] When the lactose-containing medium of this embodiment is in a solidified form in a container such as a petri dish before use, the bacterium of the present invention can be selectively grown as colonies by the culture step of this embodiment. Regarding colonies, the explanation of the first embodiment is incorporated herein by reference. Therefore, this embodiment may include a step of detecting the colonies (detection step) after the culture step, and may also include a step of isolating the bacterium of the present invention from the colonies (separation step). This embodiment may also include a step of culturing the isolated bacterium of the present invention after the separation step (post-separation culture step). The culture method may be a conventional method for culturing the bacterium of the present invention.

[0076] (Culture for detecting specific Bifidobacterium species) A third aspect of the present invention is A medium for detecting Bifidobacterium longum subsp. longum NITE BP-02621, Contains lactose, Use at 38-45°C. Culture medium is.

[0077] With regard to Bifidobacterium longum subsp. longum NITE BP-02621, the explanation in the first embodiment is incorporated herein by reference.

[0078] The medium of this embodiment is described in the same manner as in the first embodiment.

[0079] The medium of this embodiment is a medium for detecting the bacterium of the present invention. "Detecting the bacterium of the present invention" may mean detecting the bacterium of the present invention by selectively growing the bacterium of the present invention while not allowing other bacteria to grow. The other bacteria are not limited as long as they can be contained in a sample to be inoculated into the medium of this embodiment and do not grow when the medium of this embodiment is used. In other words, the medium of this embodiment may be a medium for detecting the bacterium of the present invention by selectively growing the bacterium of the present invention while not allowing other bacteria to grow.

[0080] The medium of this embodiment allows the bacteria of the present invention to be selectively grown as colonies and clearly detected. Therefore, "detecting the bacteria of the present invention" may mean detecting the bacteria of the present invention by selectively growing the bacteria of the present invention as colonies. Furthermore, "detecting the bacteria of the present invention by selectively growing the bacteria of the present invention while not allowing other bacteria to grow" may mean detecting the bacteria of the present invention by selectively growing the bacteria of the present invention as colonies while not allowing other bacteria to grow as colonies.

[0081] The explanation for the first embodiment applies to the sample to be inoculated into the medium of this embodiment.

[0082] For the other bacteria, the explanation in the first embodiment is applicable.

[0083] The medium of this embodiment is used at a predetermined temperature when in use. The explanation of the predetermined temperature is as described in the first embodiment.

[0084] A method for detecting the bacterium of the present invention using the medium of this embodiment will be described in the embodiment below.

[0085] When the medium of this embodiment is in a solidified form in a container such as a petri dish before use, the bacterium of the present invention can be selectively grown as a colony. In this case, the explanation of the colony in the first embodiment is also applicable.

[0086] (Method for detecting specific Bifidobacterium bacteria) A fourth aspect of the present invention is A method for detecting Bifidobacterium longum subsp. longum NITE BP-02621, comprising: inoculating the sample into a medium containing lactose; Culturing the bacteria contained in the sample at 38 to 45°C; and Colony detection step A method comprising: is.

[0087] With regard to Bifidobacterium longum subsp. longum NITE BP-02621, the explanation in the first embodiment is incorporated herein by reference.

[0088] This embodiment includes a step of inoculating a sample into a culture medium containing lactose (inoculation step).

[0089] For the lactose-containing medium in this step, the explanation of the medium in the first embodiment is applicable.

[0090] This embodiment is a method for detecting the bacterium of the present invention. "Detecting the bacterium of the present invention" may mean detecting the bacterium of the present invention by selectively growing the bacterium of the present invention while not allowing other bacteria to grow. The other bacteria are not limited as long as they can be contained in a sample to be inoculated into a lactose-containing medium and do not grow in the culture step described below. In other words, the method of this embodiment may be a method of detecting the bacterium of the present invention by selectively growing the bacterium of the present invention while not allowing other bacteria to grow.

[0091] According to the method of this embodiment, the bacterium of the present invention can be selectively grown as a colony and clearly detected. Therefore, "detecting the bacterium of the present invention" may mean detecting the bacterium of the present invention by selectively growing the bacterium of the present invention as a colony. Furthermore, "detecting the bacterium of the present invention by selectively growing the bacterium of the present invention while not allowing other bacteria to grow" may mean detecting the bacterium of the present invention by selectively growing the bacterium of the present invention as a colony while not allowing other bacteria to grow as colonies.

[0092] The sample in this step is similar to the description in the first embodiment.

[0093] For the other bacteria, the explanation in the first embodiment is applicable.

[0094] The method for inoculating the lactose-containing medium with the sample in this step is the same as that described in the second embodiment.

[0095] This embodiment includes, after the inoculation step, a step of culturing the bacteria contained in the sample at a predetermined temperature (for example, 38 to 45° C.) (culturing step). The description of the first embodiment is applicable to the predetermined temperature. The explanation for the second embodiment applies to the culture method in this step.

[0096] This embodiment includes a step of detecting colonies of the bacterium of the present invention (detection step) after the culturing step. When the lactose-containing medium of this embodiment is solidified in a container such as a petri dish before use, the bacterium of the present invention can be selectively grown as colonies by the culturing step. Therefore, the colonies that appear can be detected. The explanation of the colonies in the first embodiment is incorporated herein by reference.

[0097] This embodiment may also include a step of isolating the bacterium of the present invention from the colony after the detection step (isolation step). This embodiment may also include a step of culturing the isolated bacterium of the present invention after the isolation step (post-isolation culture step). The culture method may be a conventional method for culturing the bacterium of the present invention. [Example]

[0098] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0099] <Test Example 1: Examination of medium components and culture temperature> The conventional medium used for culturing Bifidobacterium longum subsp. longum (conventional medium) was TOS propionic acid agar (Yakult Pharmaceutical Co., Ltd.). The test medium used was a medium with approximately the same composition as TOS propionic acid agar (Yakult Pharmaceutical Co., Ltd.) but with a different sugar content (Table 1). Specifically, the sugar content of the conventional medium was galactooligosaccharide, whereas the sugar content of the test medium was lactose. While the conventional medium uses peptone, the test medium used a form of casein enzymatic hydrolysate (Bacto® Proteose Peptone No. 3 (Gibco®, product number (catalog number): 211693)). The pH was approximately 6.3. Each medium was autoclaved at 115°C for 15 minutes. Both media were supplemented with antibiotics, namely trimethoprim (Tokyo Chemical Industry Co., Ltd.) solution (0.008 g / L) and mupirocin (Merck Millipore) solution (0.05 g / L).

[0100] [Table 1]

[0101] Colony formation was confirmed using the prepared medium by the culture method (plate smear method) according to the following procedure. The test strain was Bifidobacterium longum subsp. longum JCM1217. T (Hereafter, JCM1217 T ) and Bifidobacterium longum subsp. longum NITE BP-02621 (hereinafter referred to as NITE BP-02621) were used. Each was cultured in mMRS medium (modified MRS medium) (de Man Rogo sa and Sharp, Becton Dickinson) supplemented with 0.05% (w / v) L-cysteine ​​hydrochloride using Anaeropack (Mitsubishi Gas Chemical Co.) under anaerobic conditions for 16 hours. The culture solution was diluted with physiological saline to 10 7 100 μL of the diluted solution was smeared onto the above agar medium and cultured for 3 days under anaerobic conditions using Anaeropack (Mitsubishi Gas Chemical Company, Inc.) at five different temperatures (37°C, 40°C, 41°C, 42°C, 43°C). Colony formation was then visually evaluated.

[0102] The results are shown in Table 2. The culture conditions under which colony formation was observed are marked with a circle, and the culture conditions under which colonies were not observed are left blank. In the conventional medium, JCM1217 was able to grow at all temperatures except 43°C among the culture temperatures evaluated. T and NITE BP-02621 were confirmed to form colonies. T While no colony formation was observed at temperatures other than 37°C, growth was confirmed in NITE BP-02621 at all temperatures. Therefore, it was found that by culturing at temperatures between 40°C and 43°C using the test medium, NITE BP-02621 could be selectively cultivated from the two strains tested.

[0103] [Table 2]

[0104] <Test Example 2: Evaluation of selectivity of modified medium> It was confirmed whether the culture method used in Test Example 1 could be used to selectively culture other Bifidobacterium longum subsp. longum strains. The test strains were as shown in Table 3. The test strains were cultured in mMRS medium using Anaeropack under anaerobic conditions for 16 hours, and then diluted with saline for 10 min. 7 100 μL of the diluted solution was smeared on the above agar medium. It was cultured at 42°C for 3 days under anaerobic conditions using Anaeropack, and the presence or absence of colony formation was evaluated visually. The results are shown in Table 3. In the conventional medium, colony formation was observed in 10 of the 16 test strains (including the two strains used in Test Example 1), but in the test medium, colony formation was observed only in NITE BP-02621. These results indicate that NITE BP-02621 can be selectively cultivated by changing the medium composition from that of the conventional medium and by changing the culture temperature.

[0105] [Table 3]

Claims

1. A medium for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621, Contains lactose, When used, it is kept at 38-45°C. Culture medium.

2. A method for selectively growing Bifidobacterium longum subsp. longum NITE BP-02621, comprising: inoculating the sample into a medium containing lactose; and Cultivating the bacteria contained in the sample at 38 to 45°C A method comprising:

3. A medium for detecting Bifidobacterium longum subsp. longum NITE BP-02621, Contains lactose, When used, it is kept at 38-45°C. Culture medium.

4. A method for detecting Bifidobacterium longum subsp. longum NITE BP-02621, comprising: inoculating the sample into a medium containing lactose; Cultivating the bacteria contained in the sample at 38 to 45°C; and detecting colonies after the culturing step A method comprising:

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